CN113960024B - Lily quality visual identification method - Google Patents
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Abstract
The invention discloses a visual identification method of lily quality, which comprises the steps of uniformly mixing an extracting solution of a lily sample to be detected with gold and silver nanoclusters, standing for reaction, and realizing quick visual identification of the lily quality according to color or fluorescence intensity information of a detection system obtained after the reaction and color or fluorescence intensity response differences among different quality lily samples. Compared with the existing detection method based on chromatography and the like, the detection method has the advantages of high stability, quick response, simple and convenient operation, portable device and the like, has wide application prospect, and can be put into industrial production.
Description
Technical Field
The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a new quality visual identification method for lilies.
Background
The lily is a traditional medicinal and edible product, is rich in various components such as polysaccharide, steroid saponin, phenolic acid and the like, has the health-care effects of nourishing yin and moisturizing lung, relieving cough and diminishing inflammation, clearing away heart fire and soothing nerves and the like, and also has the drug effects of resisting cancer, resisting depression, resisting aging, enhancing body immunity and the like. The health care function and the drug effect of the lily are closely related to the quality of the lily, and the excellent quality of the lily is related to the suitable geographical environment, climatic condition and mature production and processing technology. The quality of lily in different producing areas has great difference, for example, the producing area of the medicinal lily is Hunan Longhui, longshan, xinshao, etc., and the producing area of the edible lily is Gansu Lanzhou. Lilies of different origins are only visually indistinguishable from the appearance, especially by processed lilies.
Due to different conditions such as water, soil and climate and the like of different producing areas, the quality of the lily is divided into good and bad, which is particularly characterized in that the variety and the content of active compounds contained in the lily are greatly different, so that the drug effect is very different. At present, the quality research of common lily or other traditional Chinese medicinal materials is generally realized by methods such as chromatography, near infrared spectroscopy and the like, and the methods of the large instruments have high cost, complex operation and long time consumption, can only be limited in a laboratory, cannot be accepted by the masses of people, and are difficult to popularize.
Therefore, a method which has high sensitivity, good specificity and low cost and can quickly and conveniently identify the quality of the lily is further established, and the method has great practical significance.
Disclosure of Invention
The invention mainly aims to provide a new method for visually identifying lily quality aiming at the problems and the defects in the prior art, which utilizes the combination of gold and silver nanoclusters (Ag-AuNCs) and catechin and other components in lily to generate aggregation-induced fluorescence enhancement (AIE), and realizes the visual identification of the lily quality by comparing the obviously rich fluorescence color change generated before and after reaction; the method has the advantages of simple process, environmental protection, low cost, easy popularization, industrial production and suitability for popularization and application.
In order to achieve the purpose, the invention adopts the technical scheme that:
a visualized identification method for lily quality comprises the following steps: mixing the extracting solution of the lily sample to be detected and the gold and silver nanoclusters uniformly, standing for reaction, and realizing quick visual identification of the quality of the lily according to the color or fluorescence intensity information of the detection system obtained after the reaction and the color or fluorescence intensity response difference of the lily samples with different qualities.
In the scheme, the standing reaction time is 1-5min.
In the scheme, the concentration of the gold and silver nanoclusters in the obtained detection system is 0.67-1.67 multiplied by 10 -6 mol/L; the content of Bulbus Lilii component in the detection system is 3-7mg crude drug/mL based on Bulbus Lilii crude drug introduced in the preparation process of the extractive solution.
In the scheme, in the visualized identification method for lily quality, the color information is obtained by means of visual observation or shooting equipment, and a visualized identification map library of high-quality production areas (or genuine production areas) is obtained by comparison according to the production areas and the years of lily.
Preferably, the photographing means further photographs under ultraviolet lamp irradiation.
In the scheme, a fluorescence spectrophotometer is adopted to detect fluorescence intensity; the measurement conditions include: the emission wavelength is 360-700nm, the excitation wavelength is 330-350nm, and the slit width is 5-20nm.
In the above scheme, the visualized identification method of lily quality specifically comprises the following steps:
1) Obtaining extracting solutions of lily samples to be detected in different producing areas and/or years;
2) Mixing the gold and silver nanocluster solution emitting red fluorescence with a lily extracting solution to be detected, standing for reaction, and collecting a visual fluorescence image of the obtained detection system based on the gold and silver nanocluster sensing fluorescence enhancement effect;
3) And establishing a visual identification model base based on the producing area and/or the year of the lily according to the relation between the visual fluorescent image and the producing area and/or the year of the lily, so as to realize the identification of the high-quality producing area or the high-quality year lily products.
In the above scheme, the method for establishing the visual recognition model library includes: and extracting RGB values of the visualized fluorescence images obtained according to the lily samples in different producing areas and/or years, and constructing colorimetric cards corresponding to the lily samples in different producing areas or years.
In the above scheme, the quality of the corresponding lily sample in the colorimetric card sequentially changes from pink, purple and blue according to the law from high to low
In the above scheme, the high-quality production area (or the on-site production area) is Hunan Longya, hunan Longshan, hunan New Shao or Gansu Lanzhou (the response result is close to pink); the high-quality years are 2-3 years.
In the scheme, the preparation method of the gold and silver nanocluster comprises the following steps: adding a bovine serum albumin solution into water, then sequentially adding a chloroauric acid solution and a citrate solution, adjusting the pH value to 4-6, heating in a dark water bath, adding a silver salt solution, continuously and fully reacting, centrifuging, and filtering to obtain the red-fluorescence gold and silver nanocluster solution.
In the scheme, the molar ratio of the Bovine Serum Albumin (BSA), the chloroauric acid and the silver salt is 1 (0.22-0.67) to 0.22-0.67.
In the scheme, the silver salt can be silver nitrate, silver chloride, silver acetate or the like.
In the scheme, the concentration of the gold and silver nanocluster solution is 2-5 multiplied by 10 -4 mol/L。
In the scheme, the heating temperature of the lucifugal water bath is 70-85 ℃, and the time is 3.5-4.5h.
In the scheme, the centrifugation speed is 7000-10000rpm, and the time is 5-15min; the resulting supernatant was filtered through a 0.22 μm microfiltration membrane.
In the scheme, the extraction adopts an ultrasonic alcohol extraction process, and the adopted extraction solvent is 50-100vol% ethanol water solution.
In the above scheme, the ultrasonic alcohol extraction process conditions include: pulverizing Bulbus Lilii, grinding, sieving with 50-200 mesh sieve to obtain Bulbus Lilii powder, mixing Bulbus Lilii powder and 50-100vol% ethanol water solution according to a material-to-liquid ratio of 1g.
In the scheme, the quality of the lily to be detected comprises the production place or the year of the lily, wherein the production place comprises Hunan lily, lanzhou, fujian, jiangxi, yunnan, jiangsu and the like; the years include 1 to 8 years.
In the above scheme, a 96-well plate is used for detecting lily quality, specifically as follows: accurately measuring lily extract and water with different qualities in different producing areas respectively by using a pipette gun, adding the lily extract and the water into a 96-well plate for incubation for 1 minute, accurately measuring lily extract, water and gold and silver nanocluster solutions with different qualities in different producing areas respectively by using the pipette gun under the condition of the same concentration, and adding the lily extract, the water and the gold and silver nanocluster solutions into the 96-well plate for incubation for 1 minute; then, under 365nm exciting light of an ultraviolet dark box, a smart phone (model is IPHONE XR) is used for shooting photos of fluorescence color change generated before and after lily reaction, and meanwhile, quality difference of the lily can be judged by naked eyes.
Compared with the prior art, the invention has the beneficial effects that:
1) The invention utilizes the characteristic that the gold and silver nanoclusters are combined with effective components such as catechin in lily to generate AIE effect so as to further cause fluorescence enhancement, and combines the content difference (quality difference) of the effective components such as catechin in lily in different producing areas or different years, and utilizes the fluorescence color difference (color or fluorescence intensity difference) generated by lily products with different qualities to realize the quick visual identification of the quality of the lily.
2) Compared with the traditional detection method based on chromatography, the identification method has the advantages of higher stability, quicker response, simpler and more convenient operation, more portable device and the like, can meet the use requirements of the general public, has wide application prospect, and can be put into industrial production.
Drawings
FIG. 1 is a schematic flow chart of a lily quality identification method according to the present invention;
FIG. 2 is a graph of the visualized fluorescence results of reactions before and after the reaction of lily and nano-gold and silver clusters of different origins in example 1 of the present invention; wherein (a) is an autofluorescence visualization chart of the lily before reaction, and (b) is a fluorescence visualization chart of the lily after reaction;
FIG. 3 is a graph of the visualized fluorescence results of the reactions before and after the reaction of lily and gold and silver nanoclusters of different growth years in example 2 of the present invention;
FIG. 4 is a visual fluorescence diagram of the reaction between the standard substance of effective component catechin of lily with different concentrations and the nano-gold and silver clusters in example 1;
FIG. 5 is a fluorescence spectrum intensity diagram before and after reaction of catechin and gold and silver nanoclusters in the mechanism study of the method of the present invention.
FIG. 6 is (a) fluorescence intensity and (b) linear graph after reaction of catechin standard samples with different concentrations and nano gold and silver clusters in the mechanism research of the method of the invention;
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the following examples, the preparation method of the nano gold and silver cluster solution includes the following steps: adding 0.04mL of bovine serum albumin solution with the concentration of 50mmol/L into 1.56mL of water, then sequentially adding 0.1mL of chloroauric acid solution with the concentration of 5mmol/L and 0.2mL of citrate solution with the concentration of 0.5mmol/L, uniformly mixing, adjusting the pH value to 6, heating in a dark water bath (80 ℃,4 h), adding 0.1mL of silver nitrate solution with the concentration of 5mmol/L, continuously and fully reacting (25 ℃,3 h) to obtain a red fluorescent gold and silver nano-cluster solution product (with the concentration of 2 multiplied by 10) -4 mol/L); diluting with water 100 times the volume of the product during application to obtain a concentration of 2 × 10 -6 And (3) a gold and silver nanocluster solution in mol/L.
In the following examples, catechin standards were used as supplied by Shanghai-derived Phyllobiology, inc.
In the following examples, the extraction steps of the lily sample include the following: respectively crushing and grinding 1g of lily sample, sieving with a 50-mesh sieve to obtain lily powder, mixing the lily powder and ethanol water solution (75 vol%) according to a material-liquid ratio of 1g.
Example 1
A visualized identification method of lily quality (lily producing area) comprises the following steps:
1) Nine different known production places (Hunan lily No. 1, hunan Longya lily No. 2, hunan Longshan lily No. 3, gansu Lanzhou lily No. 4, fujian Pu Tian No. 5, 8-year-old Lanzhou lily No. 6, jiangxi Cunzhou No. 7, yunnan lily No. 8, jiangsu lily No. 9, jiangxi Dabieshan No. 10; wherein the Hu nan lily 1 is not marked with a more specific producing area when purchased) to obtain a lily extracting solution;
2) Mixing the obtained lily extract with gold and silver nanocluster solution respectively, standing for reaction for 3min, and observing fluorescence color change of the lily extract before and after the reaction; the method comprises the following specific steps:
a) Adding 900 mu of LTris-HCl buffer solution (pH = 7.0) and 100 mu of lily extract solution 10-fold diluent (diluted by water) prepared in the step 1) into a 1.5mL cuvette respectively, reacting for 3min, and detecting the fluorescence property of the solution; the obtained fluorescence photo and the fluorescence picture drawn after extracting RGB value by software are shown in figure 2a;
b) Taking a 96-microporous plate with a volume of 400 μ L, and accurately measuring 10-fold diluent, 100 μ L of buffer solution of LTris-HCl and 100 μ L of 2 × 10 concentration of lily sample extractive solution from different production areas with a pipette -6 mixing the gold and silver nanocluster solution in mol/L, and standing for reaction for 3min; shooting a picture of fluorescence color change generated after lily reaction by using a smart phone (model is IPHONE XR) under 365nm exciting light of an ultraviolet dark box, and then further extracting RGB values of the corresponding picture by using software and drawing a fluorescence picture; specific results are shown in FIG. 2b; in the figure, the detection method of Blank group (Blank) includes: accurately measuring 200 mu L of LTris-HCl buffer solution and 100 mu L of 2 × 10 concentration by using a pipette -6 mixing the gold and silver nanocluster solution in mol/L, and standing for reaction for 3min; the detection method of the catechin group (Epicatechin and catechins) comprises the following steps: accurately measuring 100 μ L catechin standard sample, 100 μ L Tris-HCl buffer solution and 100 μ L of 2 × 10 -6 mixing the gold and silver nanocluster solution in mol/L, and standing for reaction for 3min.
The detection result shows that: the self fluorescence intensity of the lily extract liquid produced in different places is weak (figure 2 a), the difference of the fluorescence intensity is small, and the difference is not easy to be identified by naked eyes; however, after the gold and silver nanoclusters are added, the gold and silver nanoclusters and catechins in the lilies in different production places can promote the generation of obvious differences in fluorescence intensity (fig. 2 b) after responding to the catechins, and the obvious differences correspond to pink, purple and blue colors which are visible to naked eyes in different degrees respectively; wherein the Hunan Longya lily and the Gansu Lanzhou lily are pink (similar to the response effect of the catechin standard sample) and correspond to the production areas of the way; it is demonstrated that the three high-quality production areas of the Hunan Longya lily (serial number 2), the Hunan Longshan lily serial number 3 and the Gansu Lanzhou lily (serial number 4) can be visually identified well by naked eyes by adopting the method of the present invention.
Example 2
A visualized identification method for lily quality (lily growth years) comprises the following steps:
1) Extracting Bulbus Lilii samples from four different years (1 year, 2 years, 3 years, 8 years, lanzhou, qilihe, etc.);
2) Diluting 100 μ L of 10 times of the obtained Bulbus Lilii extractive solution (diluting with water) with 100 μ L of LTris-HCl buffer solution, 100 μ L of 2 × 10 -6 Reacting the mol/L gold and silver nanocluster solution in a 96 microporous plate with the aperture of 400 mu L for 2min, and observing fluorescence color change of the lily extracting solution to be detected before and after the reaction under excitation light of 365nm in an ultraviolet dark box; photographs of fluorescence color changes before and after lily reaction were taken using a smartphone (model number IPHONE XR), and the results are shown in fig. 3a; the software is then used to further extract the RGB values of the corresponding photograph and to render a fluorescent picture, see fig. 3b.
As can be seen, the lily extract liquid per se in different years is weak in fluorescence intensity, small in fluorescence intensity difference and not easy to identify by naked eyes; but after the reaction with the nano gold and silver clusters, macroscopic fluorescence color change is generated, and the lilies with different qualities in different years have abundant and obvious fluorescence color differences, so that the lilies in four different years in the Lanzhou region can be well visually identified by naked eyes.
Principle study
As catechin is one of important functional components of lily, the quality of the lily is influenced by the content of the catechin, meanwhile, the content of the catechin in the lily in different producing areas has larger difference, in order to explore the visual influence of the producing areas on the quality of the lily, catechin standard samples with different concentrations and lily samples in different producing areas are respectively taken for visual identification, and the method specifically comprises the following steps:
respectively adding 100 μ L of the extract at a concentration of 0-2 × 10 -3 mol/L (specifically 1X 10) -5 、2×10 -5 、3×10 -5 、1×10 -4 、2×10 -4 、3×10 -4 、5×10 -4 、1×10 -3 、1.5×10 -3 、2×10 -3 mol/L) of catechin standard sample solution with 100. Mu.L of Tris-HCl buffer solution and 100. Mu.L of 2X 10 concentration -6 mixing of mol/L nano gold and silver cluster solutionMixing uniformly, standing for reaction for 3min, and observing fluorescence color change of standard samples of catechin with different concentrations after reaction, wherein the result is shown in figure 4; in FIG. 4, the concentrations of the original (before mixing) catechin standard sample solutions corresponding to samples Nos. 1 to 6 were 2X 10 in this order -3 、1×10 -3 、3×10 -4 、1×10 -4 、3×10 -5 、1×10 -5 mol/L; the detection solution obtained from the blank group had a concentration of 2X 10 from 100. Mu.L -6 mixing the nanometer gold and silver cluster solution of mol/L with 200 mu LTris-HCl buffer solution.
As can be seen from fig. 4, as the concentration of catechin increases, after the catechin is combined with the gold and silver nanocluster solution, an obvious color change reaction occurs, and the color changes from purple which is visible to naked eyes to pink.
Fig. 5 is a fluorescence spectrum intensity diagram before and after the reaction of the catechin standard sample and the gold and silver nanoclusters, and it can be seen that the fluorescence intensity of the gold and silver nanoclusters and the lily after the response of catechin is obviously different (obviously enhanced), and the absorption peak is blue-shifted.
FIG. 6 is a graph showing the fluorescence intensity and linearity of the reaction between catechin standard samples with different concentrations and the nano-gold and silver clusters, and it can be seen that the fluorescence intensity of the gold and silver clusters is gradually increased along with the increase of the concentration of catechin and is within a certain concentration range (0.00-2 × 10) - 3 mol/L) presents a good linear relation; therefore, the gold and silver nanoclusters adopted by the invention can be combined with the effective component catechin in the lily to generate an AIE effect, so that the fluorescence is enhanced, and then the effective identification of the quality of the lily (different products or years) is promoted and realized by combining the difference between the catechins contained in the lily in different producing areas or different years.
The above embodiments are merely examples for clearly illustrating the present invention and do not limit the present invention. Other variants and modifications of the invention, which are obvious to those skilled in the art and can be made on the basis of the above description, are not necessary or exhaustive for all embodiments, and are therefore within the scope of the invention.
Claims (9)
1. A visualized identification method for lily quality is characterized by comprising the following steps: uniformly mixing an extracting solution of a lily sample to be detected with gold and silver nanoclusters, standing for reaction, collecting a visual fluorescence image of the obtained detection system based on the sensing fluorescence enhancement effect of the gold and silver nanoclusters, and realizing visual identification of the quality of the lily by using the color or fluorescence intensity response difference of the lily samples with different qualities according to the color or fluorescence intensity information of the detection system obtained after the reaction;
the color or fluorescence intensity information is the difference of fluorescence colors generated by different quality lily products.
2. The visual identification method according to claim 1, wherein the standing reaction time is 1-5min.
3. The visual identification method as claimed in claim 1, wherein the concentration of gold and silver nanoclusters in the obtained detection system is 0.67-1.67 x 10 -6 mol/L; the content of Bulbus Lilii component in the obtained detection system is 3-7mg crude drug/mL based on Bulbus Lilii crude drug introduced in the preparation process of the extractive solution.
4. The visual identification method according to claim 1, characterized in that the color information is acquired by means of visual observation or photographing equipment; and detecting fluorescence intensity information by using a fluorescence spectrophotometer.
5. The visual identification method as claimed in claim 1, wherein the preparation method of the gold and silver nanoclusters comprises the following steps: adding a bovine serum albumin solution into water, then sequentially adding a chloroauric acid solution and a citrate solution, adjusting the pH value of the obtained solution system to 4-6, heating in a dark water bath, adding a silver salt solution, continuously reacting fully, centrifuging, and filtering to obtain a red-fluorescence gold-silver nanocluster solution.
6. The visual identification method of claim 5, wherein the molar ratio of the bovine serum albumin, the chloroauric acid and the silver salt is 1 (0.22 to 0.67): (0.22 to 0.67).
7. The visual identification method as claimed in claim 1, wherein the extraction solution is prepared by ultrasonic alcohol extraction process, and the extraction solvent is 50-100vol% ethanol water solution.
8. The visual identification method as claimed in claim 7, wherein the ultrasonic alcohol extraction process conditions adopt a material-liquid ratio of 1g to 5-10mL, a temperature of 35-40 ℃ and a time of 1.1-1.5h.
9. The visual identification method according to claim 1, wherein the lily samples to be tested comprise different producing areas and/or years, wherein the producing areas comprise Hunan, lanzhou, fujian, jiangxi, yunnan or Jiangsu; the years include 1 to 8 years.
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| CN202111137833.9A CN113960024B (en) | 2021-09-27 | 2021-09-27 | Lily quality visual identification method |
| US17/903,222 US20230102022A1 (en) | 2021-09-27 | 2022-09-06 | Method for visually identifying quality of lilii bulbus |
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| CN109738406B (en) * | 2019-01-03 | 2021-06-18 | 中南民族大学 | Method for quantitatively determining catechins |
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